307
Clinica Chimica Acta, 87 (1978) 307-318 0 Elsevier/North-Holland Biomedical Press
CGA 8985
AUTOMATED
TESTS FOR THE ASSESSMENT
LYNN NYE a,*, MARY J. ANDERSON and G.C. FORREST b
OF THYROID
b, C. DAWES b, J. LANDON
FUNCTION
a
a Department of Chemical Pathology, St. Bartholomew’s Hospital, London EC1 and b Technicon Methods and Standards Laboratory, London EC1 (U.K.) (Received
January
25th, 1978)
Summary Fully automated methods have been developed for the determination of thyroxine and triiodothyronine levels, antibodies to thyroglobulin and the assessment of thyroid hormone binding proteins in serum, using a continuous flow radioimmunoassay system. In addition the feasibility of a partially automated assay for thyrotrophin levels has been demonstrated. These employ AutoAnalyzer modules and antibodies covalently linked to a magnetisable solid phase support. Separation of bound and free antigen is achieved by applying an external magnetic field. The system currently operates at a rate of 30 samples/h and requires only 10 minutes incubation since it is not necessary to reach equilibrium. The results are similar to those obtained by conventional manual techniques, however the precision is improved and operator error eliminated.
Introduction Thyroid disease is one of the commonest endocrine abnormalities. The measurement of thyroxine (T4) together with some estimation of thyroid hormone binding proteins, followed by calculation of the free thyroxine index (FTI) is commonly employed as a screening test. If the FTI is normal, or just outside of the normal range, total triiodothyronine (T3) estimations are of value in the assessment of suspected hyperthyroidism and thyrotrophin (TSH) estimations in the assessment of suspected hypothyroidism [l] . It is well known that antibodies to thyroglobulin are present in the serum of patients with Hashimoto’s thyroiditis [2] and a quantitative test for such antibodies would also be of value in assessing thyroid function. With conventional manual procedures it may take several days to complete * Correspondence should be addressed to: Dr. L. Nye, Department of Immunology, Medical School, 40-50 Tottenham Street. London WlP 9PG. U.K.
Middlesex Hospital
308
these tests and the precision obtained may be poor. Use of fully automated systems, such as those described in this study, can enable completion of all these analyses within a working day, with good precision. In addition, the number of requests for radioimmunoassays such as those employed in thyroid function testing is rapidly increasing and, in large laboratories, will necessitate the introduction of fully automated systems. Both discrete and continuous flow systems have been described [ 3,4]. Materials and methods Manual assays Radioimmunoassays. Total T4 levels were measured by the method of Nye and her colleagues [ 51 and total T3 levels by that of Chopra and his group [6]. For these assays S-anilino-1-naphthalene sulphonic acid (ANS) was employed to block binding sites on endogenous thyroxine binding globulin (TBG). The antibody bound fractions were precipitated by the addition of polyethylene glycol in the T4 assay and by co-precipitation with donkey anti-rabbit serum in the T3 assay. Anti-thyroglobulin antibodies (TgAb). These were measured by the method of Roitt and Doniach [7] in which positive sera agglutinate tanned sheep red blood cells coated with thyroglobulin (Tg). Thyroid hormone uptake test (THUT). This was performed using the Thyopac 3 kit (Radiochemical Centre, Amersham) according to the manufacturer’s introductions. Automated
assays
Reagents Buffer. 0.05 M phosphate buffer, pH 7.4, containing 0.25% Tween 20, 0.1% sodium azide and 0.25% bovine serum albumin was employed for all assays. Barbital buffer is usually recommended for the T4 and T3 assays but is not necessary [ 51. Standard solutions. For the T4 and T3 assays these were as described for the manual procedures. For the THUT assay, 1000 ml of serum from normal donors was pooled, tested for Australia antigen, passed through a 0.45 pm Millipore filter and stored in aliquots at -20°C. TSH standard solutions were prepared in horse serum. Labelled hormones. 1251-T4 and ‘*‘I-T3, with specific activities of approximately 1800 pCi/pg, were prepared by the method of Weeke and &skov [8] and diluted for use to contain 1 ng and 0.5 ng/ml, respectively. 1251-Tg and ‘*‘ITSH with specific activities of 70 and 180 @i/pg respectively were prepared by the method of Hunter and Greenwood [9] and diluted for use to contain 20 and 1.5 ng/ml, respectively. Antibodies. Anti-T3 and anti-T4 were obtained by immunising sheep with carbodiimide linked T,:BSA or T,:BSA conjugates. The anti-T3 serum was supplied by Dr. J.G. Ratcliffe (Radioimmunoassay Unit, Stobhill General Hospital, Glasgow) and anti-T4 and donkey anti-rabbit immunoglobulin sera by
309
Dr. T.G. Merrett (Rast Allergy Unit, Benenden Chest Hospital, Benenden, Kent). Rabbit anti-human TSH was obtained by immunisation with highly purified human TSH. Preparation of the magnetisable particles (MP). The assays described employed MP consisting of iron oxide (Fe,04) embedded in a cellulose matrix [lo], however a variety of other particles, including ones based on autoactive polymers, have proved equally useful. After grinding to a suitable size (l-2 pm) the particles were sedimented using a magnet and the supernatant aspirated. The particles were then washed with 5-6 1 cold distilled water until the pH was reduced to approximately 7.2 and finally with buffer, prior to coupling [ 111 to the gamma globulin fractions from the appropriate antiserum or normal serum, prepared by precipitation with anhydrous sodium sulphate (0.18 g/ml). Coupling was achieved by suspending the activated magnetic particles in buffer and adding the partially purified globulin fractions followed by vertical rotation for 3 days at 4°C. The solid phase was then sedimented on the magnet and the supernatant aspirated. Finally, the particles were washed at room temperature with buffer, and stored at a concentration of 1 g/10 ml in phosphate buffer at 4°C. Under these storage conditions the binding capacity did not significantly decrease over a period of several months. Serum samples. For the Tq and THUT assays blood was collected from: (a) 120 healthy euthyroid subjects who were not taking oral contraceptives, (b) 19 euthyroid subjects who were more than 32 weeks pregnant, (c) 15 hyperthyroid patients, (d) 11 hypothyroid patients. For the T3 and TgAb assays, samples were selected from sera sent to the laboratory for routine analyses. Varying amounts of standard T3 were added to some of the former.
Equipment This is a Technicon system described previously [ 121. The control module is a sequence programmer which has two independent functions: one controlling the addition of reagents, the other the separation and counting sequence. Initiation of the assay sequence by the operator starts a series of events. Test samples are aspirated into the system at a rate of 30/h with a sample : wash ratio of 1 : 2, a segment of labelled antigen is added to the serum sample and, after mixing in a short coil, a segment of solid phase antibody solution is added. The reaction mixtures are incubated for 10 min at room temperature during passage through a glass mixing coil. The separation and counting sequence is activated by a particle detector which initiates a series of events when the opaque reaction segments interrupt the beam. Two electromagnets are switched on and the solid-phase particles are retained on the side of the tube at the points of contact with the poles of the first magnet. After 60 set, when sufficient buffer has passed to ensure adequate washing, the first magnet is switched off and the particles are collected at the second magnet, where they are washed for a further 15 sec. During this time the supernatant and wash solutions flow into a spill proof waste container via a valve. The direction of the valve is reversed when the second magnet is de-ener-
310
gised, so that the solid phase is released and passed into a flow-through gamma counter where the radioactivity is counted for 80 sec.
cell in a
Principle of the THUT assay Several manual THUT tests have been employed to provide an indication of the unoccupied sites on the thyroid hormone binding proteins. These usually involve incubation of serum samples (containing the binding proteins) with 12’I-T3 and a non-specific adsorbent for the unbound labelled hormone. Initially red cells and later various resins were employed as the non-specific adsorbent. In the Thyopac 3 kit, Sephadex G-25 is used for this purpose and the amount of radioactivity remaining in the supernatant is directly related to the initial number of available binding sites on the carrier proteins. The automated THUT assay is based on a similar principle. 12’I-T3 (or 1251-T4) distributes between the binding proteins, predominantly thyroxine binding globulin (TBG), and a constant amount of anti-T3 (or anti-T4) covalently linked to magnetic particles. The 1251-T3 bound by the antibody is inversely related to the number of available binding sites. Thus, in hypothyroidism, or in situations with elevated TBG levels (such as pregnancy and patients receiving oestrogen) a larger proportion of the label will be bound by the endogenous
W-anti
T3
or MP-anti T4
MP-thyroglobulin
l25*_T or 1251-T3 4
Serum containing thyroid hormone, binding proteins
\ Serum containing antibodies to thyroglobulin
125 I-thyroglobulin
Fig. 1. (a) Principle of the automated THUT assay. The amount of labelled hormone bound by the antibody linked to the magnetic particles (MP) will be inversely proportional to the number of available binding sites in the serum sample. (b) Principle of the automated TgAb assay. The amount of 1 251-thyroglobulin bound to the MP wilJ be proportional to the amount of anti-thyroglobulin antibody in the serum sample.
311
binding proteins than when normal serum is present. In hyperthyroidism, or in people with low TBG levels (due to old age or androgen therapy) or in people taking drugs (such as phenytoin or salicylates) which bind to the TBG binding sites, the reverse is found (Fig. la). Principle
of the test for antibodies
to thyroglobulin
Serum samples are incubated with lz51-Tg and Tg covalently bound to magnetic particles. When anti-Tg antibodies are present in the sample the situation occurs where one binding site will bind to the Tg on the magnetic particles and the other to the 1251-Tg. Thus the amount of “‘1-Tg bound to the magnetic particle will be proportional to the amount of anti-Tg in the sample, and is less than 2% of the total radioactivity in the absence of antibody (Fig. lb). Semi-au toma ted TSH assay
Preliminary experiments were performed to investigate the feasibility of a fully automated TSH assay. However, the lo-min incubation period employed was not sufficient to obtain adequate binding and a pre-incubation step was necessary. Standards were incubated with 12’I-TSH and anti-TSH, at a final concentration of 1 : 200 000 for 6 h at room temperature. The antibody bound and free fractions were then separated by aspiration into the automated system and reaction for 10 min with donkey anti-rabbit immunoglobulin bound to magnetisable particles. Calculations
For the T4, T3 and TSH assays the antibody bound fraction was expressed as a percentage of the binding (C!,) obtained for the zero standard (Ci/Co X 100). The best cubic curve was computed for the standards by the method of least squares and the amount of hormone in the test samples was interpolated using a suitable computer programme. For the anti-Tg antibody assay, results were expressed as the number of counts per second bound to the magnetic particles. For the THUT assay, the uptake values were expressed as a percentage of a normal pooled serum (test/standard X 100). The free thyroxine index (FTI) was calculated from the T4 and THUT values. When the latter is measured by the automated method: FTI
=
Ta X THUT 100
Whereas, when Thyopac FTI=A
THUT
3 values are employed:
X100
Results Automated
T4 assay
The progress of the reaction between 1251-T4 and the solid phase antibody was assessed as a function of incubation times ranging from 2 to 25 min. It was found that 85% of the binding had occurred after 10 min. Non-specific binding
312
25
50
100
200 300 400
nmol/l T4 Fig. 2. Range of values for 10 T4 standard curves run on 10 consecutive days.
TABLE I THYROXINE Q.C.
ASSAY
PRECISION
Within assay * Mean
S.D.
(nmoI/U (n = 10)
(nmoI/I)
Low Normal High
30.4 91.6 186.0
2.4 2.9 9.0
7.6 3.1 4.8
B.C.
Between assay I * Mean
S.D.
C.V. (%)
(nmoI/I) (n = 10)
(nmoI/U
Low Normal High
25.9 94.5 194.0
1.9 4.3 8.5
B.C.
Between assay II * *
Low Normal High
Mean
S.D.
(nmoI/l) (n = 10)
(nmoI/U
29.8 89.6 272.1
3.3 5.3 20.4
* Pooled sera. ** Commercial B.C. sera (HyIand).
C.V. (a)
7.7 4.5 4.4
C.V. (%)
Quoted range
11.0 6.0 7.5
28.343.7 75.7-114 202-308
313 TABLE
II
THYROXINE
ASSAY:
CROSS-REACTIVITY
AT
50%
BINDING __-___
Cross-reactivity Manual
assay
(%) Automated
assay _
39
D-Thyroxine Tetraiodothyroacetic
acid
3,5.3’-Triiodo-L-thyronine
3,5-Diodo-Gtyrosine
2.2
(T3)
3.5,3’-Triiodothyroacetic 3-Iodo-L-tyrosine
9.3
(TETRAC) acid
(TRIAC)
(MIT) (DIT)
120 38 1.6
0.3
2.8
0
0
0
0
was assessed by substituting solid phase to which normal sheep gamma-globulin had been linked. After 10 min (the incubation time finally employed in the system) non-specific binding was less than 2% of the total radioactivity. The T4 standard curve (Fig. 2) shows the range of values for 10 standard curves run on 10 consecutive days and Table I the “within” and “between” assay coefficients of variation for three different pooled sera with high, normal and low T4 values and for three commercially available control sera (Q-PAK Thyroid Function Controls, Hyland Division, Travenol Laboratories Inc., California, U.S.A.), which were also assayed on 10 consecutive days. The results are regarded as satisfactory and the values for the commercial quality control sera lie within the range quoted by the manufacturers. Similar T4 results were obtained when 60 serum samples, with values ranging between 28 and 273 nmol/l, were assayed by the manual and automated methods (r = 0.976). Recovery experiments, using three different serum samples to which either 100 or 200 nmol T,Jl had been added, gave recoveries which ranged between 97 and 107%. Specificity was assessed in the manual and automated T4 assays. The results summarised in Table II show significant differences despite the same antiserum being employed, thus D-thyroxine (which is not present in normal serum) cross-reacts by 39 and 120% in the manual and automated assays respectively and cross-reactivity with TRIAC and TETRAC was also greater in the automated system as compared with the manual assay. However, at the concentrations normally present in serum, none of the analogues tested will affect the measurement of T4 by either assay. Automated
T3 assay The T3 standard curve illustrated in Fig. 3 shows that the assay can be employed to distinguish between normal and elevated serum T3 levels. Similar T3 levels were obtained by the manual and automated assays for 20 serum samples and 30 serum samples to which standard T3 had been added, to give values ranging from between 5 and 25 nmol/l (r = 0.958). Automated
anti-Tgantibody
assay
Thirty serum samples which gave negative results when measured by the manual assay gave values ranging from 21 to 54 (mean 34.3 + 9.0) in the auto-
314
60 %Co 50
L
0.625
1
4
2.5 nml/l
10
4
I
40
160
T3
Fig. 3. The Tj standard curve.
counts/set bound to MP 600
500
. . .
400
i i i .. :
300
200
I . . . 0. :
.:. ..
100
h I
Normal sera (negative TRC) n=30
Positive TRC n=40
Fig. 4. Values obtained by the automated TgAb test for 30 normal serum samples and 40 samples which gave positive values by the tanned red cell haemagglutination assay.
315
mated system. Forty serum samples with manual values ranging between 1 : 20 to 1 : 5 000 000 gave values ranging between 39 and 600 (mean 214 + 136). There was little overlap between the two groups (Fig. 4) but there was no correlation between the titre obtained in the manual assay and the amount of ‘*‘I-Tg bound to the magnetic particles in the automated assay. Au toma ted THUT assay 1. Employing ‘*‘I-T3 and anti-T3.
Maximum differences between the hypothyroid and hyperthyroid ranges were obtained employing 0.5 ng per ml of ‘*‘I-T3 and 25 mg per ml of anti-T3 linked magnetisable particles. Serum THUT values of 99.7 & 8.5 (mean ? S.D.) with a range of 84 to 116 were obtained for 50 euthyroid subjects. Fig. 5 compares THUT values obtained for 85 serum samples by Thyopac 3 and the automated method. The range obtained by the latter was much wider (57 to 194) than by Thyopac 3 (75 to 151) due, in large part, to nine samples from clinically thyrotoxic patients. When these samples were excluded there was a good correlation (r = 0.966). THUT values by Thyopac 3 or the automated method, for the hypothyroid, hyperthyroid and pregnant groups, were employed together with total TJ levels to calculate the FTI (Fig. 6). The pregnant group, whose T4 levels were either
Automated THUT. 210
l
o Hypothyroid
.
190
l
170
l
l
Pregnant
A Normal
.
150
Hyperthyroid
.
.
‘.’ 130
110
90
70
50
,
I
I
70
90
110
1 130
150
170
Thyopac 3 Fig. 5. Comparison assay.
of THUT values obtained for 85 serum samples by Thyopac 3 and the automated
316 Free
thyroxine index (FTI) 0
T
Thyopac 3
q Hyperthyroid
Automated THUT.
q Automated THUT.
300
200 150
_______-----
100
60 -
50 250
____________ L
:... .
Clinica Chimica Acta, 87 (1978) 307-318 0 Elsevier/North-Holland Biomedical Press
CGA 8985
AUTOMATED
TESTS FOR THE ASSESSMENT
LYNN NYE a,*, MARY J. ANDERSON and G.C. FORREST b
OF THYROID
b, C. DAWES b, J. LANDON
FUNCTION
a
a Department of Chemical Pathology, St. Bartholomew’s Hospital, London EC1 and b Technicon Methods and Standards Laboratory, London EC1 (U.K.) (Received
January
25th, 1978)
Summary Fully automated methods have been developed for the determination of thyroxine and triiodothyronine levels, antibodies to thyroglobulin and the assessment of thyroid hormone binding proteins in serum, using a continuous flow radioimmunoassay system. In addition the feasibility of a partially automated assay for thyrotrophin levels has been demonstrated. These employ AutoAnalyzer modules and antibodies covalently linked to a magnetisable solid phase support. Separation of bound and free antigen is achieved by applying an external magnetic field. The system currently operates at a rate of 30 samples/h and requires only 10 minutes incubation since it is not necessary to reach equilibrium. The results are similar to those obtained by conventional manual techniques, however the precision is improved and operator error eliminated.
Introduction Thyroid disease is one of the commonest endocrine abnormalities. The measurement of thyroxine (T4) together with some estimation of thyroid hormone binding proteins, followed by calculation of the free thyroxine index (FTI) is commonly employed as a screening test. If the FTI is normal, or just outside of the normal range, total triiodothyronine (T3) estimations are of value in the assessment of suspected hyperthyroidism and thyrotrophin (TSH) estimations in the assessment of suspected hypothyroidism [l] . It is well known that antibodies to thyroglobulin are present in the serum of patients with Hashimoto’s thyroiditis [2] and a quantitative test for such antibodies would also be of value in assessing thyroid function. With conventional manual procedures it may take several days to complete * Correspondence should be addressed to: Dr. L. Nye, Department of Immunology, Medical School, 40-50 Tottenham Street. London WlP 9PG. U.K.
Middlesex Hospital
308
these tests and the precision obtained may be poor. Use of fully automated systems, such as those described in this study, can enable completion of all these analyses within a working day, with good precision. In addition, the number of requests for radioimmunoassays such as those employed in thyroid function testing is rapidly increasing and, in large laboratories, will necessitate the introduction of fully automated systems. Both discrete and continuous flow systems have been described [ 3,4]. Materials and methods Manual assays Radioimmunoassays. Total T4 levels were measured by the method of Nye and her colleagues [ 51 and total T3 levels by that of Chopra and his group [6]. For these assays S-anilino-1-naphthalene sulphonic acid (ANS) was employed to block binding sites on endogenous thyroxine binding globulin (TBG). The antibody bound fractions were precipitated by the addition of polyethylene glycol in the T4 assay and by co-precipitation with donkey anti-rabbit serum in the T3 assay. Anti-thyroglobulin antibodies (TgAb). These were measured by the method of Roitt and Doniach [7] in which positive sera agglutinate tanned sheep red blood cells coated with thyroglobulin (Tg). Thyroid hormone uptake test (THUT). This was performed using the Thyopac 3 kit (Radiochemical Centre, Amersham) according to the manufacturer’s introductions. Automated
assays
Reagents Buffer. 0.05 M phosphate buffer, pH 7.4, containing 0.25% Tween 20, 0.1% sodium azide and 0.25% bovine serum albumin was employed for all assays. Barbital buffer is usually recommended for the T4 and T3 assays but is not necessary [ 51. Standard solutions. For the T4 and T3 assays these were as described for the manual procedures. For the THUT assay, 1000 ml of serum from normal donors was pooled, tested for Australia antigen, passed through a 0.45 pm Millipore filter and stored in aliquots at -20°C. TSH standard solutions were prepared in horse serum. Labelled hormones. 1251-T4 and ‘*‘I-T3, with specific activities of approximately 1800 pCi/pg, were prepared by the method of Weeke and &skov [8] and diluted for use to contain 1 ng and 0.5 ng/ml, respectively. 1251-Tg and ‘*‘ITSH with specific activities of 70 and 180 @i/pg respectively were prepared by the method of Hunter and Greenwood [9] and diluted for use to contain 20 and 1.5 ng/ml, respectively. Antibodies. Anti-T3 and anti-T4 were obtained by immunising sheep with carbodiimide linked T,:BSA or T,:BSA conjugates. The anti-T3 serum was supplied by Dr. J.G. Ratcliffe (Radioimmunoassay Unit, Stobhill General Hospital, Glasgow) and anti-T4 and donkey anti-rabbit immunoglobulin sera by
309
Dr. T.G. Merrett (Rast Allergy Unit, Benenden Chest Hospital, Benenden, Kent). Rabbit anti-human TSH was obtained by immunisation with highly purified human TSH. Preparation of the magnetisable particles (MP). The assays described employed MP consisting of iron oxide (Fe,04) embedded in a cellulose matrix [lo], however a variety of other particles, including ones based on autoactive polymers, have proved equally useful. After grinding to a suitable size (l-2 pm) the particles were sedimented using a magnet and the supernatant aspirated. The particles were then washed with 5-6 1 cold distilled water until the pH was reduced to approximately 7.2 and finally with buffer, prior to coupling [ 111 to the gamma globulin fractions from the appropriate antiserum or normal serum, prepared by precipitation with anhydrous sodium sulphate (0.18 g/ml). Coupling was achieved by suspending the activated magnetic particles in buffer and adding the partially purified globulin fractions followed by vertical rotation for 3 days at 4°C. The solid phase was then sedimented on the magnet and the supernatant aspirated. Finally, the particles were washed at room temperature with buffer, and stored at a concentration of 1 g/10 ml in phosphate buffer at 4°C. Under these storage conditions the binding capacity did not significantly decrease over a period of several months. Serum samples. For the Tq and THUT assays blood was collected from: (a) 120 healthy euthyroid subjects who were not taking oral contraceptives, (b) 19 euthyroid subjects who were more than 32 weeks pregnant, (c) 15 hyperthyroid patients, (d) 11 hypothyroid patients. For the T3 and TgAb assays, samples were selected from sera sent to the laboratory for routine analyses. Varying amounts of standard T3 were added to some of the former.
Equipment This is a Technicon system described previously [ 121. The control module is a sequence programmer which has two independent functions: one controlling the addition of reagents, the other the separation and counting sequence. Initiation of the assay sequence by the operator starts a series of events. Test samples are aspirated into the system at a rate of 30/h with a sample : wash ratio of 1 : 2, a segment of labelled antigen is added to the serum sample and, after mixing in a short coil, a segment of solid phase antibody solution is added. The reaction mixtures are incubated for 10 min at room temperature during passage through a glass mixing coil. The separation and counting sequence is activated by a particle detector which initiates a series of events when the opaque reaction segments interrupt the beam. Two electromagnets are switched on and the solid-phase particles are retained on the side of the tube at the points of contact with the poles of the first magnet. After 60 set, when sufficient buffer has passed to ensure adequate washing, the first magnet is switched off and the particles are collected at the second magnet, where they are washed for a further 15 sec. During this time the supernatant and wash solutions flow into a spill proof waste container via a valve. The direction of the valve is reversed when the second magnet is de-ener-
310
gised, so that the solid phase is released and passed into a flow-through gamma counter where the radioactivity is counted for 80 sec.
cell in a
Principle of the THUT assay Several manual THUT tests have been employed to provide an indication of the unoccupied sites on the thyroid hormone binding proteins. These usually involve incubation of serum samples (containing the binding proteins) with 12’I-T3 and a non-specific adsorbent for the unbound labelled hormone. Initially red cells and later various resins were employed as the non-specific adsorbent. In the Thyopac 3 kit, Sephadex G-25 is used for this purpose and the amount of radioactivity remaining in the supernatant is directly related to the initial number of available binding sites on the carrier proteins. The automated THUT assay is based on a similar principle. 12’I-T3 (or 1251-T4) distributes between the binding proteins, predominantly thyroxine binding globulin (TBG), and a constant amount of anti-T3 (or anti-T4) covalently linked to magnetic particles. The 1251-T3 bound by the antibody is inversely related to the number of available binding sites. Thus, in hypothyroidism, or in situations with elevated TBG levels (such as pregnancy and patients receiving oestrogen) a larger proportion of the label will be bound by the endogenous
W-anti
T3
or MP-anti T4
MP-thyroglobulin
l25*_T or 1251-T3 4
Serum containing thyroid hormone, binding proteins
\ Serum containing antibodies to thyroglobulin
125 I-thyroglobulin
Fig. 1. (a) Principle of the automated THUT assay. The amount of labelled hormone bound by the antibody linked to the magnetic particles (MP) will be inversely proportional to the number of available binding sites in the serum sample. (b) Principle of the automated TgAb assay. The amount of 1 251-thyroglobulin bound to the MP wilJ be proportional to the amount of anti-thyroglobulin antibody in the serum sample.
311
binding proteins than when normal serum is present. In hyperthyroidism, or in people with low TBG levels (due to old age or androgen therapy) or in people taking drugs (such as phenytoin or salicylates) which bind to the TBG binding sites, the reverse is found (Fig. la). Principle
of the test for antibodies
to thyroglobulin
Serum samples are incubated with lz51-Tg and Tg covalently bound to magnetic particles. When anti-Tg antibodies are present in the sample the situation occurs where one binding site will bind to the Tg on the magnetic particles and the other to the 1251-Tg. Thus the amount of “‘1-Tg bound to the magnetic particle will be proportional to the amount of anti-Tg in the sample, and is less than 2% of the total radioactivity in the absence of antibody (Fig. lb). Semi-au toma ted TSH assay
Preliminary experiments were performed to investigate the feasibility of a fully automated TSH assay. However, the lo-min incubation period employed was not sufficient to obtain adequate binding and a pre-incubation step was necessary. Standards were incubated with 12’I-TSH and anti-TSH, at a final concentration of 1 : 200 000 for 6 h at room temperature. The antibody bound and free fractions were then separated by aspiration into the automated system and reaction for 10 min with donkey anti-rabbit immunoglobulin bound to magnetisable particles. Calculations
For the T4, T3 and TSH assays the antibody bound fraction was expressed as a percentage of the binding (C!,) obtained for the zero standard (Ci/Co X 100). The best cubic curve was computed for the standards by the method of least squares and the amount of hormone in the test samples was interpolated using a suitable computer programme. For the anti-Tg antibody assay, results were expressed as the number of counts per second bound to the magnetic particles. For the THUT assay, the uptake values were expressed as a percentage of a normal pooled serum (test/standard X 100). The free thyroxine index (FTI) was calculated from the T4 and THUT values. When the latter is measured by the automated method: FTI
=
Ta X THUT 100
Whereas, when Thyopac FTI=A
THUT
3 values are employed:
X100
Results Automated
T4 assay
The progress of the reaction between 1251-T4 and the solid phase antibody was assessed as a function of incubation times ranging from 2 to 25 min. It was found that 85% of the binding had occurred after 10 min. Non-specific binding
312
25
50
100
200 300 400
nmol/l T4 Fig. 2. Range of values for 10 T4 standard curves run on 10 consecutive days.
TABLE I THYROXINE Q.C.
ASSAY
PRECISION
Within assay * Mean
S.D.
(nmoI/U (n = 10)
(nmoI/I)
Low Normal High
30.4 91.6 186.0
2.4 2.9 9.0
7.6 3.1 4.8
B.C.
Between assay I * Mean
S.D.
C.V. (%)
(nmoI/I) (n = 10)
(nmoI/U
Low Normal High
25.9 94.5 194.0
1.9 4.3 8.5
B.C.
Between assay II * *
Low Normal High
Mean
S.D.
(nmoI/l) (n = 10)
(nmoI/U
29.8 89.6 272.1
3.3 5.3 20.4
* Pooled sera. ** Commercial B.C. sera (HyIand).
C.V. (a)
7.7 4.5 4.4
C.V. (%)
Quoted range
11.0 6.0 7.5
28.343.7 75.7-114 202-308
313 TABLE
II
THYROXINE
ASSAY:
CROSS-REACTIVITY
AT
50%
BINDING __-___
Cross-reactivity Manual
assay
(%) Automated
assay _
39
D-Thyroxine Tetraiodothyroacetic
acid
3,5.3’-Triiodo-L-thyronine
3,5-Diodo-Gtyrosine
2.2
(T3)
3.5,3’-Triiodothyroacetic 3-Iodo-L-tyrosine
9.3
(TETRAC) acid
(TRIAC)
(MIT) (DIT)
120 38 1.6
0.3
2.8
0
0
0
0
was assessed by substituting solid phase to which normal sheep gamma-globulin had been linked. After 10 min (the incubation time finally employed in the system) non-specific binding was less than 2% of the total radioactivity. The T4 standard curve (Fig. 2) shows the range of values for 10 standard curves run on 10 consecutive days and Table I the “within” and “between” assay coefficients of variation for three different pooled sera with high, normal and low T4 values and for three commercially available control sera (Q-PAK Thyroid Function Controls, Hyland Division, Travenol Laboratories Inc., California, U.S.A.), which were also assayed on 10 consecutive days. The results are regarded as satisfactory and the values for the commercial quality control sera lie within the range quoted by the manufacturers. Similar T4 results were obtained when 60 serum samples, with values ranging between 28 and 273 nmol/l, were assayed by the manual and automated methods (r = 0.976). Recovery experiments, using three different serum samples to which either 100 or 200 nmol T,Jl had been added, gave recoveries which ranged between 97 and 107%. Specificity was assessed in the manual and automated T4 assays. The results summarised in Table II show significant differences despite the same antiserum being employed, thus D-thyroxine (which is not present in normal serum) cross-reacts by 39 and 120% in the manual and automated assays respectively and cross-reactivity with TRIAC and TETRAC was also greater in the automated system as compared with the manual assay. However, at the concentrations normally present in serum, none of the analogues tested will affect the measurement of T4 by either assay. Automated
T3 assay The T3 standard curve illustrated in Fig. 3 shows that the assay can be employed to distinguish between normal and elevated serum T3 levels. Similar T3 levels were obtained by the manual and automated assays for 20 serum samples and 30 serum samples to which standard T3 had been added, to give values ranging from between 5 and 25 nmol/l (r = 0.958). Automated
anti-Tgantibody
assay
Thirty serum samples which gave negative results when measured by the manual assay gave values ranging from 21 to 54 (mean 34.3 + 9.0) in the auto-
314
60 %Co 50
L
0.625
1
4
2.5 nml/l
10
4
I
40
160
T3
Fig. 3. The Tj standard curve.
counts/set bound to MP 600
500
. . .
400
i i i .. :
300
200
I . . . 0. :
.:. ..
100
h I
Normal sera (negative TRC) n=30
Positive TRC n=40
Fig. 4. Values obtained by the automated TgAb test for 30 normal serum samples and 40 samples which gave positive values by the tanned red cell haemagglutination assay.
315
mated system. Forty serum samples with manual values ranging between 1 : 20 to 1 : 5 000 000 gave values ranging between 39 and 600 (mean 214 + 136). There was little overlap between the two groups (Fig. 4) but there was no correlation between the titre obtained in the manual assay and the amount of ‘*‘I-Tg bound to the magnetic particles in the automated assay. Au toma ted THUT assay 1. Employing ‘*‘I-T3 and anti-T3.
Maximum differences between the hypothyroid and hyperthyroid ranges were obtained employing 0.5 ng per ml of ‘*‘I-T3 and 25 mg per ml of anti-T3 linked magnetisable particles. Serum THUT values of 99.7 & 8.5 (mean ? S.D.) with a range of 84 to 116 were obtained for 50 euthyroid subjects. Fig. 5 compares THUT values obtained for 85 serum samples by Thyopac 3 and the automated method. The range obtained by the latter was much wider (57 to 194) than by Thyopac 3 (75 to 151) due, in large part, to nine samples from clinically thyrotoxic patients. When these samples were excluded there was a good correlation (r = 0.966). THUT values by Thyopac 3 or the automated method, for the hypothyroid, hyperthyroid and pregnant groups, were employed together with total TJ levels to calculate the FTI (Fig. 6). The pregnant group, whose T4 levels were either
Automated THUT. 210
l
o Hypothyroid
.
190
l
170
l
l
Pregnant
A Normal
.
150
Hyperthyroid
.
.
‘.’ 130
110
90
70
50
,
I
I
70
90
110
1 130
150
170
Thyopac 3 Fig. 5. Comparison assay.
of THUT values obtained for 85 serum samples by Thyopac 3 and the automated
316 Free
thyroxine index (FTI) 0
T
Thyopac 3
q Hyperthyroid
Automated THUT.
q Automated THUT.
300
200 150
_______-----
100
60 -
50 250
____________ L
:... .
